Neutrophil Extracellular Traps in ST-Segment Elevation Myocardial Infarction

Background Interleukin-6-receptor inhibition with tocilizumab improves myocardial salvage in patients with ST-segment elevation myocardial infarction (STEMI). Reduced levels of neutrophil extracellular traps (NETs), which consist of nuclear material studded with proteins released upon neutrophil activation, might contribute to this effect. Objectives The purpose of this study was to evaluate the effect of tocilizumab on NETs and investigate the association between NETs and myocardial injury in patients with STEMI. Methods In the ASSAIL-MI study, 199 patients with STEMI were randomized to tocilizumab or placebo during percutaneous coronary intervention. In this substudy, we analyzed blood levels of the NET markers double-stranded deoxyribonucleic acid (dsDNA), myeloperoxidase-DNA, and citrullinated histone 3 (H3Cit) at admission and after 24 hours and 3 to 7 days. In a subgroup of patients, we assessed regulation of transcripts related to the formation of NETs. We also investigated associations between NET markers and the myocardial salvage index (MSI). Results All NET markers were lower in the tocilizumab group than in the placebo group at 3 to 7 days (all P < 0.04). Several NET-related pathways were downregulated in the tocilizumab group. The beneficial effect of tocilizumab on the MSI seemed to be partly dependent on reduction of NETs (structural equation modeling: 0.05, P = 0.001 [dsDNA] and 0.02, P = 0.055 [H3Cit]). Patients with NETs in the 3 lowest quartiles had higher MSI than patients in quartile 4 (10.9 [95% CI: 4.0-15.0] [dsDNA] and 8.9 [95% CI: 2.0-15.9] [H3Cit], both P = 0.01). Conclusions NETs were reduced by tocilizumab and associated with myocardial injury. The effect of tocilizumab on MSI might be mediated through reduced NETs. (ASSessing the Effect of Anti-IL-6 Treatment in Myocardial Infarction: The ASSAIL-MI Trial [ASSAIL-MI]; NCT03004703)

I schemia reperfusion injury is respon- sible for up to 50% of the final infarct size after ST-segment elevation myocardial infarction (STEMI). 1 The pathophysiological mechanism involves activation of innate immunity, but treatment options remain elusive. 2 In the ASSessing the effect of Anti-IL-6 treatment in Myocardial Infarction (ASSAIL-MI) trial, we showed that a single dose of the interleukin (IL)-6 receptor inhibitor tocilizumab attenuated myocardial injury in patients with acute STEMI.Tocilizumab infusion started prior to and continued alongside primary percutaneous coronary intervention with an infusion time of 1 hour.Tocilizumab significantly increased myocardial salvage index (MSI), 3 ie, the fraction of the ischemic myocardium (area at risk) rescued from necrosis. 4There was also a numerically, although not statistically significant, difference in infarct size quantified by cardiac magnetic resonance imaging and peak levels of troponin T between the treatment groups.Subsequent analyses showed that treatment with tocilizumab was associated with a fall in neutrophil cell count as well as attenuated inflammatory potential of the residual neutrophils, including degranulation. 5 Neutrophils produce neutrophil extracellular traps (NETs), 6 which are filamentous, thread-like structures of double-stranded deoxyribonucleic acid (dsDNA) twined around histones like citrullinated histone 3 (H3Cit).2][13] Interestingly, targeting NETs with DNAse 1 has been shown to accelerate ex vivo tissue plasminogen activator-induced thrombolysis. 14 have previously shown that level of the NET marker dsDNA was associated with infarct size and death in patients with STEMI. 15,16Our aim in the present study was to explore how tocilizumab influenced circulating NET levels in the setting of ischemia reperfusion injury in STEMI patients.Because the IL-6 pathway is thought to stimulate NETosis, 17 we hypothesized that treatment with the IL-6 receptor antagonist tocilizumab would be associated with reduced circulating levels of NET markers, again contributing to myocardial salvage.

METHODS
ETHICAL CONSIDERATIONS.The trial protocol is approved by the regional ethics committee (REK Sør-Øst 2016/1223), and all participants provided written informed consent.The safety of the trial was monitored by an independent Data and Safety Monitoring Board.The trial adhered to the principles of the Declaration of Helsinki and followed the guidelines for good clinical practice.Prior to enrolling participants, the trial was registered at ClinicalTrials.gov,NCT03004703.
STUDY DESIGN.We analyzed data from patients enrolled in the ASSAIL-MI trial between March 16, 2017, and February 13, 2020.The design of ASSAIL-MI has been published previously. 3The trial was conducted at 3 high-volume percutaneous coronary intervention centers in Norway and designed as a randomized, double-blind, placebo-controlled trial.Kindberg et al  4.8% (dsDNA), 5.3% (MPO-DNA), and 11.9% (H3Cit).
See Supplemental Table 1 for technical specifications on the MPO-DNA and H3Cit enzyme-linked immunosorbent assay.
Total RNA was isolated from BD PAXgene Blood RNA tubes (BD Biosciences) using MagMAX for Stabilized Blood Tubes RNA Isolation Kit (Invitrogen) following the manufacturer's instructions.RNA isolation, RNA sequencing, and bioinformatic analysis were performed as previously described in detail by Huse et al. 5 Neutrophil-imputed genes 5 were imported into Rstudio (v.2022.12.0þ353).We performed an over-representation analysis on the "Neutrophil Extracellular Trap Formation pathway" (hsa04613) from the Kyoto Encyclopaedia of Genes and Genomes (KEGG).The analysis involved preranking of genes based on their log2fold difference between the tocilizumab and placebo group.We utilized the cluster Profiler (v. 4   2).
TOCILIZUMAB TREATMENT AND NETs: MODULATION OF NET-RELATED PATHWAYS IN NEUTROPHILS.Enrichment analysis of neutrophil-imputed genes revealed an overrepresentation of genes belonging to the "Neutrophil Extracellular Trap Formation" pathway at 3 to 7 days (Figure 2A).There was a decrease in the transcriptional levels of histone H3, its citrullinated form, citH3, and histone deacetylases (HDAC), all related to NET formation, in patients treated with tocilizumab.However, there was also a decrease in Tocilizumab and NETs in STEMI Siglec-9 expression, suggested to inhibit neutrophil activation.Additionally, there was an increased transcriptional expression of H2A, H2B, and H4, all components of core histones, and, to some extent, Rac (Figure 2A).Of the 89 neutrophil-imputed genes detected in the NETs formation pathway (Supplemental Figure 2), 14 genes were significantly regulated by tocilizumab at an individual gene level, of which the majority were lower in the tocilizumabtreated group (Figure 2B).Nevertheless, our results support a causal association between NET reduction and increased MSI.The reduction at the transcriptional levels of genes related to NET formation suggests that NETs indeed were downregulated by tocilizumab.We have previously shown that the beneficial effect of tocilizumab in ASSAIL-MI might be mediated through reduced numbers of neutrophils and functional attenuation and inhibition of degranulation in these cells. 5In the present study, we extend these findings by showing decreased neutrophil transcriptional levels of histone H3, its citrullinated form, citH3, and histone deacetylases, all related to NETs formation.Interestingly,   Although we find lower levels of NET markers in STEMI patients treated with tocilizumab, we have previously reported increased levels of H3Cit and no changes in dsDNA with tocilizumab treatment in patients with non-ST-elevation myocardial infarction (NSTEMI). 23The reason for this discrepancy is not clear but could reflect differences in infarct size, differences in time from symptom onset, and differences in comorbidities and coronary pathophysiology between patients with STEMI and NSTEMI. 24one could argue that the statistical analyses were prone to a type II error.We and others have previously shown similar associations between dsDNA and measurements of cardiac function, 7,15 and levels of H3Cit have been associated with cardiovascular outcome after STEMI. 25There are several proposed mechanisms for how NETs contribute to the myocardial injury in STEMI.9][30] Activated toll-like receptors may also activate the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in cardiomyocytes, 31 which further activates the inflammatory cascade of the IL1-IL6 pathway. 17Moreover, the pathogenic interactions between platelets, NETs, and endothelial cells are well established in various disorders 8,32 and may be relevant in the setting of STEMI.Also, NETs and H3Cit can stimulate the IL-1-IL-6 pathway, 33,34 at least partly through nucleotide-binding oligomerization domain-like receptor protein 3 activation in macrophages, potentially representing a vicious cycle in STEMI.Thus, whereas IL-1 is upstream for IL-6 in this cascade and tocilizumab does not influence IL-1 levels, 35,36 the present study shows that targeting IL-6 signaling influences NET formation, potentially contributing to the beneficial effects of tocilizumab in these patients.As tocilizumab inhibits both transand classical IL-6 signaling, of which the latter may at least partly have some anti-inflammatory effects, 37 forthcoming studies could also test the effects of specific inhibition of IL-6 trans-signaling in STEMI patients.
The relationship between serum NET markers and myocardial injury must be interpreted with caution.
There are other sources of circulating dsDNA beyond NETs, including necrosis in the area of the myocardial scar.Animal studies have nevertheless repeatedly reported that infarct size can be reduced by reducing NETs. 9,10,38The association between NETs and myocardial injury support NETs as possible treatment targets in patients with STEMI.
STUDY LIMITATIONS.This study has several limitations, among which are the modest sample size, the relatively small myocardial infarctions, and the

2 4
TOCILIZUMAB TREATMENT AND NETs: SERUM MARKERS.Serum levels of dsDNA, MPO-DNA, and H3Cit levels from baseline to 6 months in the 2 randomized groups are shown in Figure1.Levels of all markers were similar between the 2 treatment groups at baseline.However, levels of dsDNA and MPO-DNA were lower in the tocilizumab group than in the placebo group at 24 hours and 3 to 7 days (dsDNA: 354 vs 367 ng/ml[24 hours] and 337 vs 387 ng/ml [3-7 days]; MPO-DNA: 0.099 vs 0.107 OD [24 hours] and 0.092 vs 0.101 OD [3-7 days], all P # 0.04) (Figure1).While Kindberg et alJ A C C : A D V A N C E S , V O L . 3 , N O .9 , 2 0Tocilizumab and NETs in STEMIS E P T E M B E R 2 0 2 4 : 1 0 1 1 9 3unaffected at 24 hours, H3Cit levels were lower in the tocilizumab than in the placebo group at 3 to 7 days (1.36 vs 2.13 ng/ml, P ¼ 0.012).The change from baseline to 3 to 7 days differed between the groups for dsDNA levels (tocilizumab À7 ng/ml vs placebo 35 ng/ ml, P < 0.001), but not for MPO-DNA and H3Cit.After 3 and 6 months, there were no differences between the treatment groups for any of the markers.NET MARKERS: INTERCORRELATION AND ASSOCIATION WITH NEUTROPHIL COUNTS AND PLATELETS.dsDNA and MPO-DNA intercorrelated weakly at baseline and 24 h (r ¼ 0.162 and 0.214, both P < 0.03).MPO-DNA and H3Cit correlated only at baseline (r ¼ 0.224, P < 0.002), while dsDNA and H3Cit correlated only after 3 to 7 days (r ¼ 0.187, P ¼ 0.001).All NET markers were moderately positively correlated to corresponding neutrophil cell count, and there was no correlation to platelet count (Supplemental Table

FIGURE 1
FIGURE 1 Time Profiles of NET Markers by Treatment Arms

FIGURE 2
FIGURE 2 Transcriptional Regulation of NETosis-Related Pathway by Tocilizumab CONTRIBUTION FROM NETs ON THE EFFECT OF TOCILIZUMAB ON MYOCARDIAL SALVAGE.In ASSAIL-MI, the effect of tocilizumab on the MSI was assessed by linear regression adjusted for time from symptom onset. 3We expanded the model with the addition of NET markers separately.As shown in Table 2, lower coefficient values were demonstrated when adding NET markers to the model.The coefficient for the effect of tocilizumab on MSI was 0.06 when only adjusting for time from symptom onset.When adjusting for H3Cit, the coefficient dropped to 0.04.After adjustment for dsDNA, the coefficient dropped further to À0.002.Structural equation modeling was used to establish the direct and indirect pathways for the effect of tocilizumab on MSI when including H3Cit and dsDNA as mediators.

Figure 3
shows how the indirect effect of tocilizumab on the MSI through dsDNA and H3Cit at 3 to 7 days is 0.05 (P ¼ 0.001) and 0.02 (P ¼ 0.055), respectively, illustrating that attenuated NET levels may contribute to the effect of tocilizumab in ASSAIL-MI.NETs AND MYOCARDIAL INJURY AS ASSESSED BY CARDIAC MAGNETIC RESONANCE.In a linear regression model adjusted for age, sex, and tocilizumab treatment, dsDNA and H3Cit at 3 to 7 days were significantly associated with the MSI (Table 3).Patients in Q1-3 of dsDNA and H3Cit had larger MSIs (b ¼ 10.9 [95% CI: 3.2-18.6],P < 0.006 [dsDNA] and b ¼ 8.9 [95% CI: 2.0-15.9],P ¼ 0.01 [H3Cit]).There was no association between MPO-DNA and the MSI.Also, in a linear regression model adjusted for age, sex, and tocilizumab treatment (Table 4), dsDNA and H3Cit levels were both associated with infarct size as assessed by cardiac magnetic resonance imaging after 3 to 7 days and 6 months.Based on the visualization of quartile graphs (Supplemental Figure 3), we dichotomized dsDNA and H3Cit between quartile 3 and 4. Patients in the lowest quartiles of dsDNA (#392 ng/ml) and H3Cit (#3.2 ng/ml) had smaller

FIGURE 3
FIGURE 3 Structural Equation Modeling of Effect Pathways of Tocilizumab Tocilizumab and NETs in STEMI infarct sizes at 3 to 7 days and 6 months than patients in Q4, as shown in Figure 4. Linear regression analyses adjusted for age, sex, and tocilizumab treatment demonstrated the same pattern for the dichotomized NET markers (b ¼ À0.07, 95% CI: À0.01 to À0.032, P < 0.001 [dsDNA] and b ¼ À0.05; 95% CI À0.078 to À0-018, P ¼ 0.002 [H3Cit] at 3 to 7 days, while b ¼ À0.06, 95% CI: À0.085 to À0.026, P < 0.001 [dsDNA] and b ¼ À0.04, 95% CI: À0.067 to À0.014, P ¼ 0.003 [H3Cit] after 6 months).Levels of MPO-DNA were not associated with infarct size.Microvascular obstruction in percent of the left ventricular volume was also significantly lower in Q1-3 vs Q4 for dsDNA (P < 0.0001) and H3Cit (P ¼ 0.004) (Figure 5).In a logistic regression model adjusted for age, sex, and tocilizumab treatment, Q1-3 of dsDNA and H3Cit were associated with the absence of microvascular obstruction with an OR of 0.42 (95% CI: 0.19-0.92,P ¼ 0.03) (dsDNA) and 0.48 (95% CI: 0.24-0.97,P ¼ 0.04) (H3Cit).DISCUSSION In this substudy of the ASSAIL-MI trial, we show that tocilizumab reduced NET markers in the acute phase of STEMI (Central Illustration).The ASSAIL-MI trial demonstrated that tocilizumab increased the MSI, and our findings suggest that part of this beneficial effect of tozilizumab could be mediated through reduced formation of NETs.Low levels of the NET markers dsDNA and H3Cit were independently associated with higher MSI, smaller infarct size, and less pronounced microvascular obstruction.The findings highlight the significance of NETs in STEMI and suggest that targeted therapy against IL-6 signaling could attenuate NET formation.Tocilizumab reduced serum levels of all NET markers in this STEMI cohort.Mediation analysis done with structural equation modeling indicated that part of the beneficial effect of tocilizumab on the MSI in the ASSAIL-MI trial could be mediated through NETs.As there may be other mediators of the effect between tocilizumab and MSI that are not included in our model, the results must be reviewed with caution.

HDAC
inhibition has been shown to inhibit NETs formation induced by activated platelets, 20 and downregulation of HDAC by tocilizumab could be involved in the mechanisms by which tocilizumab attenuate NETs formation in STEMI patients.Moreover, HDAC inhibition has been suggested to attenuate myocardial ischemia reperfusion injury, 21 further supporting a beneficial role for the tocilicumab-mediated downregulation of HDAC in STEMI.At present, however, the mechanisms by which tocilizumab attenuate NETs formation are not clear.Based on the RNA seq data and the only moderate correlation of NET markers with neutrophil counts, the mechanisms seem to reflect more than just a downregulation of numbers of circulating neutrophils.However, we cannot exclude that the reduced levels of NET markers in the treatment group could involve the restraining effect tocilizumab on the extravascular pool of neutrophil cells, preventing the cells to enter the circulation 22 and thereby reducing potential NETs release.

FIGURE 4 4
FIGURE 4 Association Between Dichotomized NET Markers and Infarct Size
.6.2) package and performed the analysis with the enrichKEGG function, considering results with an adjusted P value <0.05 as statistically sig- representation analysis results were presented with the pathview function.Visual presentation of differentially regulated neutrophil-imputed genes, determined by a t-test P value <0.05, belonging to the NETs formation pathway was procured with the ComplexHeatmap (v.2.14.0) package.MSI (in %) was calculated as the difference between area at risk and infarct size, divided by area at risk Â 100.Microvascular obstruction was defined as areas with absence of gadolinium enhancement within the infarct zone.The area was manually traced and reported as percentage of the left ventricular mass.tions with the MSI, infarct size, and microvascular obstruction.We adjusted the models for the covariates age, sex, and tocilizumab treatment.We chose not to adjust for inflammatory parameters or troponin, as these are closely linked to NETs and infarct size.The level of statistical significance was set to 2-sided P < 0.05.All statistical analyses were performed on STATA v.17 SE (StataCorp LLC), except for RNA sequencing data analysis, which was performed on R version 4.2.1.RESULTSSTUDY POPULATION.A total of 199 patients were enrolled in ASSAIL-MI.The majority were men (85%), and the mean age was 61 years.One in 3 had no known prior disease, and only 6% had established cardiovascular disease (Table1).

TABLE 3
NET Markers and the MSI All variables are measured as continuous variables.Bold indicates P value < 0.05.a Adjusted for age, sex and tocilizumab treatment.dsDNA ¼ double-stranded deoxyribonucleic acid; H3Cit ¼ citrullinated histone 3; MSI ¼ myocardial salvage index; NET ¼ neutrophil extracellular trap.

TABLE 4
All variables are measured as continuous variables.Bold indicates P value < 0.05.a Adjusted for age, sex and tocilizumab treatment.Infarct size as % of left ventricle.dsDNA ¼ double-stranded deoxyribonucleic acid; H3Cit ¼ citrullinated histone 3; NET ¼ neutrophil extracellular trap.